Cyclophosphamide in Combination with Immune Therapeutics

ABSTRACT

The present invention relates to methods of treating a cancer and in particular, a B-cell derived cancer, using a lymphocytotoxic but hematopoeitic cell sparing high-dose pulsed amount of an oxazaphosphorine drug, either alone, or in combination with immune therapeutics such as, for example, monoclonal antibodies that selectively bind B-cell specific antigens.

BACKGROUND OF THE INVENTION

Cyclophosphamide and especially high-dose cyclophosphamide (for example,50 mg/kg/day×4 days) has been used for the treatment of certainautoimmune diseases such as, for example, severe aplastic anemia. Low tointermediate doses of cyclophosphamide have also been used incombination chemotherapy for treating certain cancers and appears towork either by stopping the growth of cancer cells or by killing thecancer cells. However, usually remaining cancer cells, especially cancerstem cells, are able to divide, thereby leading to a relapse of thecancer.

More recently, immune-based therapeutics, for example, monoclonalantibodies targeted selectively to tumor specific antigens have beendeveloped for treatment of various types of cancers. However, immunetolerance appears to be the major barrier to the effectiveness of mostimmune-based therapeutics. It has been reported that immune toleranceresults largely from the integration of multiple regulatory mechanismsthat have evolved to prevent the development of immunity to tumorspecific antigens, which are perceived as self antigens instead offoreign antigens.

Therefore, there is a need to identify therapies or agents which may beused to break immune tolerance which severely limits the use ofimmune-based therapeutics in the treatment of cancer.

SUMMARY OF THE INVENTION

This invention is based, at least on part, on the discovery that,high-dose cyclophosphamide is effective in breaking immune tolerancewhich presents an obstacle in the use of various immune basedtherapeutics in the treatment of various types of cancers. The presentinvention provides methods of treating various cancers and, inparticular, B-cell derived cancers, using high-dose pulsed amount of anoxazaphosphorine drug such as, for example, cyclophosphamide, eitheralone or in combination with other non-myeloablative therapies, such as,for example, monoclonal antibodies that selectively bind B-cell specificantigens such as, for example, CD-20 and CD-22.

In one aspect of the present invention, a method of eliminating orsubstantially reducing non-Hodgkin's lymphoma in a subject is provided.The method includes administering a lymphocytotoxic but hematopoeiticstem cell sparing high-dose pulsed amount of an oxazaphosphorine drug tothe subject, such that the subject's immune system reconstitutes withoutstem cell transplantation, thereby to eliminate or substantially reducenon-Hodgkin's lymphoma in the subject.

In another aspect of the present invention, a method for eliminating orsubstantially reducing Hodgkin's disease in a subject is provided. Themethod includes administering a lymphocytotoxic but hematopoeitic stemcell sparing high-dose pulsed amount of an oxazaphosphorine drug to thesubject, such that the subject's immune system reconstitutes withoutstem cell transplantation, thereby to eliminate or substantially reduceHodgkin's lymphoma in the subject.

In yet another aspect of the present invention, a method for eliminatingor substantially reducing chronic lymphocytic leukemia in a subject isprovided. The method includes administering a lymphocytotoxic buthematopoeitic stem cell sparing high-dose pulsed amount of anoxazaphosphorine drug to the subject, such that the subject's immunesystem reconstitutes without stem cell transplantation, thereby toeliminate or substantially reduce chronic lymphocytic leukemia in thesubject.

In a further aspect of the present invention, a method for eliminatingor substantially reducing mantle cell lymphoma in a subject is provided.The method includes administering a lymphocytotoxic but hematopoeiticstem cell sparing high-dose pulsed amount of an oxazaphosphorine drug tothe subject, such that the subject's immune system reconstitutes withoutstem cell transplantation, thereby to eliminate or substantially reducemantle cell lymphoma in the subject.

In a further aspect of the present invention, a method for eliminatingor substantially reducing multiple myeloma in a subject is provided. Themethod includes administering a lymphocytotoxic but hematopoeitic stemcell sparing high-dose pulsed amount of an oxazaphosphorine drug to thesubject, such that the subject's immune system reconstitutes withoutstem cell transplantation, thereby to eliminate or substantially reducemultiple myeloma in the subject.

In one or more aspects of the present invention, a method foreliminating or substantially reducing non-Hodgkin's lymphoma, Hodgkin'slymphoma, chronic lymphocytic leukemia, mantle cell lymphoma or multiplemyeloma in a subject includes administering a lymphocytotoxic buthematopoeitic stem cell sparing high-dose pulsed amount of anoxazaphosphorine drug to the subject, such that the subject's immunesystem reconstitutes without stem cell transplantation, andadministering an effective dose of a monoclonal antibody thatselectively binds a B-cell specific antigen. A monoclonal antibody usedin the methods of the invention may selectively bind an antigen chosenfrom: CD3d, CD5, CD6, CD9, CD19, CD20, CD21, CD22, CD23, CD24, CD27,CD28, CD37, CD38, CD40, CD45, CD46, CD48, CD53, CD69, CD70, CD72, CD73,CD79a, CD79b, CD80, CD81, CD83, CD85a, CD85d, CD85e, CD85h, CD85i,CD85j, CD85k, CD86, CD96, CD98, CD100, CD121b, CD124, CD127, CD132,CD150, CD152, CD154, CD157, CD166, CD169, CD179a, CD179b, CD180, CD185,CD196, CD197, CD205, CDw210a, CD213a1, CD257, CD267, CD268, CD269,CD274, CD275, CD276, CD278, CD279, CD300a, CD300c, CD307, CD314, CD316,CD317, CD319, CD320, CDw327, or CD331.

In some methods according to the present invention, a method foreliminating or substantially reducing non-Hodgkin's lymphoma, Hodgkin'slymphoma, chronic lymphocytic leukemia, mantle cell lymphoma or multiplemyeloma in a subject includes administering a lymphocytotoxic buthematopoeitic stem cell sparing high-dose pulsed amount of anoxazaphosphorine drug to the subject, such that the subject's immunesystem reconstitutes without stem cell transplantation, andadministering an effective dose of an antibody that selectively bindsCD-20, thereby to eliminate or substantially reduce non-Hodgkin'slymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, mantle celllymphoma or multiple myeloma in the subject.

In other methods according to the present invention, a method foreliminating or substantially reducing non-Hodgkin's lymphoma, Hodgkin'slymphoma, chronic lymphocytic leukemia, mantle cell lymphoma or multiplemyeloma in a subject includes administering a lymphocytotoxic buthematopoeitic stem cell sparing high-dose pulsed amount of anoxazaphosphorine drug to the subject, such that the subject's immunesystem reconstitutes without stem cell transplantation, andadministering an effective dose of an antibody that selectively bindsCD-22, thereby to eliminate or substantially reduce non-Hodgkin'slymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, mantle celllymphoma or multiple myeloma in the subject.

In some other embodiments, a lymphocytotoxic but hematopoeitic cellsparing high-dose pulsed amount of an oxazaphosphorine drug used in themethods described herein is between 100 mg/kg and 200 mg/kg,administered daily from 1 to 7 days. In other embodiments, alymphocytotoxic but hematopoeitic cell-sparing high-dose pulsed amountof an oxazaphosphorine drug is between 25 mg/kg and 100 mg/kg,administered daily for 4 consecutive days. In some embodiments, alymphocytotoxic but hematopoeitic stem cell sparing high-dose pulsedamount of an oxazaphosphorine drug is administered to the subject for 4days. In other embodiments, a lymphocytotoxic but hematopoeitic stemcell sparing high-dose pulsed amount of an oxazaphosphorine drug is 50mg/kg/day, administered for 4 days.

In various aspects of the methods of the present invention, theoxazaphosphorine drug is selected from the group consisting ofcyclophosphamide, ifosfamide, perfosfamide, trophosphamide(trofosfamide), or a pharmaceutically acceptable salt, solvate, prodrugand metabolite thereof. In some embodiments, a oxazaphosphorine drugused in the methods described herein is cyclophosphamide or apharmaceutically acceptable salt or metabolite thereof.

In still another aspect, the oxazaphosphorine drug is lyophilizedcyclophosphamide or a pharmaceutically acceptable salt, solvate,prodrug, or metabolite thereof.

In some embodiments of the present invention, an effective amount of amonoclonal antibody that selectively binds a B-cell specific antigen isbetween about 100 mg/m² to about 200 mg/m². In other embodiments of thepresent invention, an effective amount of a monoclonal antibody thatselectively binds a B-cell specific antigen is between about 200 mg/m²to about 300 mg/m², or between about 300 mg/m² to about 400 mg/m². In aparticular embodiment, an effective amount of a monoclonal antibody thatselectively binds a B-cell specific antigen is about 375 mg/m².

In one embodiment, a lymphocytotoxic hematopoeitic cell sparinghigh-dose pulsed amount of an oxazaphosphorine drug such as, forexample, 50 mg/Kg of cyclophosphamide administered daily for 4consecutive days, is administered to a subject having non-Hodgkin'slymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, mantle celllymphoma or multiple myeloma, before the administration of an effectiveamount of a monoclonal antibody that selectively binds a B-cell specificantigen.

In another embodiment, a lymphocytotoxic hematopoeitic cell sparinghigh-dose pulsed amount of an oxazaphosphorine drug such as, forexample, 50 mg/Kg of cyclophosphamide administered daily for 4consecutive days, is administered to a subject having non-Hodgkin'slymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, mantle celllymphoma or multiple myeloma, after the administration of an effectiveamount of a monoclonal antibody that selectively binds a B-cell specificantigen.

In yet another embodiment, an effective amount of a monoclonal antibodythat selectively binds a B-cell specific antigen is administered to asubject having non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia or multiple myeloma both before and afteradministration of a lymphocytotoxic hematopoeitic cell sparing high-dosepulsed amount of an oxazaphosphorine drug such as, for example, 50 mg/Kgof cyclophosphamide administered daily for 4 consecutive days.

In some embodiments of the various aspects of the methods of the presentinvention, a subject having non-Hodgkin's lymphoma or Hodgkin's lymphomaor chronic lymphocytic leukemia or mantle cell lymphoma or multiplemyeloma is further administered additional agents. Exemplary additionalagents include, for example, hematopoeitic growth factors such aspegfilgrastin.

Also encompassed by this disclosure is a kit for treating non-Hodgkin'slymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, mantle celllymphoma or multiple myeloma including: (a) a plurality of doses of alymphocytotoxic but hematopoetic cell-sparing high-dose pulsed amount ofa oxazaphosphorine drug; (b) a plurality of doses of an effective amountof one or more monoclonal antibodies that selectively bind a B-cellspecific antigen; and (c) instructions for treating non-Hodgkin'slymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, mantle celllymphoma or multiple myeloma using one or more doses of theoxazaphosphorine drug and one or more doses of one or more monoclonalantibodies that selectively bind a B-cell specific antigen, where thenon-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocyticleukemia, mantle cell lymphoma or multiple myeloma disorder is treatedwithout the need for stem cell transplantation.

In certain embodiments of the methods of the invention, the inventionincludes administering an effective amount of Mesna.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery thata lymphocytotoxic high-dose pulsed amount of an oxazaphosphorine drugsuch as cyclophosphamide, can be used for breaking immune tolerance in asubject, which presents a major obstacle in the use ofimmunotherapeutics such as, monoclonal antibodies, in the treatment ofvarious cancers and in particular, B-cell derived lymphomas. Suchlymphomas are generally regarded as incurable with conventionaltherapies. Although, they may be responsive to conventional cytotoxicchemotherapy and radiation therapy, it appears that any remaining cancercells and especially, cancer stem cells can result in a relapse of thecancer.

The present invention provides a method for eliminating or substantiallyreducing lymphomas including non-Hodgkin's lymphoma, Hodgkin's lymphoma,chronic lymphocytic leukemia, mantle cell lymphoma and multiple myelomain a subject by administering a lymphocytoxic but hematopoeiticstem-cell sparing high-dose pulsed amount of an oxazaphosphorine drugsuch as, for example, cyclophosphamide, such that the subject's immunesystem reconstitutes without stem cell transplantation.

In some embodiments, the present invention provides a method foreliminating or substantially reducing non-Hodgkin's lymphoma, Hodgkin'slymphoma, chronic lymphocytic leukemia, mantle cell lymphoma or multiplemyeloma in a subject by administering a lymphocytoxic but hematopoeiticstem-cell sparing high-dose pulsed amount of an oxazaphosphorine drugsuch as, for example, cyclophosphamide, such that the subject's immunesystem reconstitutes without stem cell transplantation, andadministering an effective amount of a monoclonal antibody thatselectively binds a B-cell specific antigen. It is understood that suchantibodies are particularly effective in depleting B-cells includingcancer stem cells which usually express B-cell specific antigens,thereby to eliminate or substantially reduce the lymphoma and incidenceof a relapse.

One or more monoclonal antibodies that selectively bind a B-cellspecific antigen may be administered either before the administration ofa lymphocytoxic but hematopoeitic stem-cell sparing high-dose pulsedamount of an oxazaphosphorine drug such as, for example,cyclophosphamide, or one or more of antibodies that selectively bind aB-cell specific antigen may be administered after the administration ofa lymphocytoxic but hematopoeitic stem-cell sparing high-dose pulsedamount of an oxazaphosphorine drug such as, for example,cyclophosphamide. In some embodiments, one or more monoclonal antibodiesthat selectively bind a B-cell specific antigen are administered to asubject both prior to and subsequent to the administration of alymphocytoxic but hematopoeitic stem-cell sparing high-dose pulsedamount of an oxazaphosphorine drug such as, for example,cyclophosphamide.

In a particular embodiment, an effective amount of a monoclonal antibodythat selectively binds a B-cell specific antigen such as, for example,rituximab, is administered to a subject having non-Hodgkin's lymphoma,Hodgkin's lymphoma, chronic lymphocytic leukemia, mantle cell lymphomaor multiple myeloma, prior to the administration of a lymphocytoxic buthematopoeitic stem-cell sparing high-dose pulsed amount of anoxazaphosphorine drug such as, for example, cyclophosphamide, therebyresulting in a synergistic effect. For example, a synergistic effect mayresult from the sensitization of B-cells associated with a lymphoma tocytotoxic agents, where the B-cells are otherwise resistant to suchagents, using a monoclonal antibody which selectively binds a B-cellspecific antigen, and subsequently exposing the sensitized B-cells to acytotoxic agent, e.g., a lymphocytotoxic but hematopoeitic cell sparinghigh-dose pulsed amount of an oxazaphosphorine drug. Accordingly, insome embodiments, a synergistic effect can be obtained by sensitizingB-cells by using a monoclonal antibody that selectively binds a B-cellspecific antigen and subsequently exposing them to a lymphocytotoxic buthematopoeitic cell sparing high-dose pulsed amount of anoxazaphosphorine drug.

In some embodiments of the present invention, additional agents and inparticular agents which facilitate hematopoeitic stem cell growth suchas, for example, filgrastim and pegfilgrastin, are administered to asubject following the administration of a lymphocytoxic buthematopoeitic stem-cell sparing high-dose pulsed amount of anoxazaphosphorine drug such as, for example, cyclophosphamide.

In some aspects, the methods of the invention may further compriseadministering an effective amount of Mesna.

I. Definitions

In order that the present disclosure may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The term “an oxazaphosphorine drug” refers to a class of drugs which actas alkylating agents and cause immunoablation. They are generally highlycytotoxic and are often used as chemotherapeutic agents. Examples ofoxazaphosphorine drugs include cyclophosphamide, ifosfamide,perfosfamide, trophosphamide (trofosfamide), and pharmaceuticallyacceptable salts, solvates, prodrugs and metabolites thereof. In someembodiments, an oxazaphosphorine drug used in the methods describedherein is cyclophosphamide, which is sold under common trade-namesincluding PROCYTOX®, CYTOXAN® and NEOSAR®. Cyclophosphamide is convertedto 4-hydroxycyclophosphamide and its tautomer aldophosphamide in theliver and is cytotoxic to cells that express low levels of the enzymealdehyde dehydrogenase, for example, NK cells and T and B lymphocytes.Ifosfamide (MITOXANA®) is a structural analog of cyclophosphamide andits mechanism of action is considered to be identical or substantiallysimilar to that of cyclophosphamide. Perfosfamide(4-hydroperoxycyclophosphamide) and trophosphamide are also alkylatingagents which are structurally related to cyclophosphamide. For example,Perfosfamide alkylates DNA, thereby inhibiting DNA replication and RNAand protein synthesis.

As used herein, the phrase “high-dose pulsed amount of anoxazaphosphorine drug” refers to a non-myeloablative amount of anoxazaphosphorine drug such as, for example, cyclophosphamide, which isimmunoablative, upon single or multiple dose administration to a subject(such as a human patient suffering from non-Hodgkin's lymphoma,Hodgkin's lymphoma, chronic lymphocytic leukemia, mantle cell lymphomaor multiple myeloma), thereby resulting in a substantial reduction in orcomplete elimination of mature circulating lymphocytes in the subject.In some embodiments, administration of a non-myeloablative amount ofcyclophosphamide results in treating, preventing, curing, delaying,reducing the severity of, ameliorating at least one symptom ofnon-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemiaor multiple myeloma, or prolonging the survival of the subject beyondthat expected in the absence of such administration. In someembodiments, “high-dose pulsed amount of an oxazaphosphorine drug”refers to a dose of cyclophosphamide administered to a subject in needthereof, which results in eliminating or substantially reducing thenumber of circulating lymphocytes in the subject, including those whichare associated with immune tolerance associated with cancer, whilesparing the hematopoeitic progenitor stem cells. For example, in someembodiments, “high-dose pulsed amount of an oxazaphosphorine drug” is a50 mg/kg/day dose of an oxazaphosphorine drug such as, for example,cyclophosphamide, administered to a subject in need thereof for 4consecutive days.

The terms “eliminating,” “substantially reducing,” “treating,” and“treatment,” as used herein, refer to therapeutic or preventativemeasures described herein. The methods of “eliminating or substantiallyreducing” employ administration to a subject having non-Hodgkin'slymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, mantle celllymphoma or multiple myeloma, a lymphocytotoxic non-myeloablative amountof an oxazaphosphorine drug such as, for example, cyclophosphamide,either alone or in combination with other non-myeloablative therapies,such as to prevent, cure, delay, reduce the severity of, or ameliorateone or more symptoms of non-Hodgkin's lymphoma, Hodgkin's lymphoma,chronic lymphocytic leukemia, mantle cell lymphoma or multiple myeloma,thereby to prolong the survival of a subject beyond that expected in theabsence of such treatment. In some embodiments, the term “eliminating”refers to a complete remission of a cancer, e.g., non-Hodgkin'slymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, mantle celllymphoma or multiple myeloma in a subject treated using the methodsdescribed herein.

The term “hematopoeitic progenitor stem cell,” as used herein refers toany type of cell of the hematopoeitic system, including, but not limitedto, undifferentiated cells such as hematopoeitic stem cells andprogenitor cells, which are capable of reconstituting the immune systemfollowing administration of a lymphocytotoxic non-myeloablative amountof cyclophosphamide to a subject identified using the methods describedherein.

The terms “immunoablation” and “immunoablative,” as used herein, referto severe immunosuppression using a high-dose (i.e., lymphocytotoxicnon-myeloablative amount) of cyclophosphamide, for example, 50 mg/kg×4days of cyclophosphamide, which leads to substantial reduction in orelimination of the population of circulating lymphocytes, including forexample, NK cells and B and T lymphocytes Immunoablation, as describedherein, results in complete or substantially complete reduction in cellsresponsible for immune tolerance.

The term “lymphocytotoxic,” as used herein, refers to completeelimination of or substantial reduction in the number of circulatinglymphocytes, including those associated with immune tolerance in asubject following administration of a high-dose (i.e., lymphocytotoxicnon-myeloablative amount) of a oxazaphosphorine drug, such as, forexample, 50 mg/kg×4 days of cyclophosphamide. The term“lymphocytotoxic,” includes killing of those immune cells bycyclophosphamide which express low levels of the enzyme aldehydedehydrogenase.

The term “non-myeloablative,” as used herein, refers to a property of acompound such as, for example, an oxazaphosphorine drug such ascyclophosphamide, whereby the compound does not have a cytotoxic effecton myeloid cells, for example, hematopoeitic progenitor stem cells. Insome embodiments, a non-myeloablative agent used in the methodsdescribed herein has a cytotoxic effect on the circulating maturelymphocytes (e.g., NK cells, and T and B lymphocytes) while sparing theprogenitor cells, e.g., hematopoeitic progenitor stem cells that arecapable of reconstituting the immune system. In some embodiments, anon-myeloablative agent used in the methods of the invention kills cellswhich express low levels of the enzyme aldehyde dehydrogenase (e.g., NKcells and B and T lymphocytes) while sparing cells which express high orresistant levels of the enzyme aldehyde dehydrogenase (e.g.,hematopoeitic progenitor stem cells).

The terms “B lymphocyte” and “B cell,” as used interchangeably herein,are intended to refer to any cell within the B cell lineage as early asB cell precursors, such as pre-B cells B220⁺ cells which have begun torearrange Ig VH genes and up to mature B cells and even plasma cellssuch as, for example, plasma cells which are associated with multiplemyeloma. The term “B-cell,” also includes a B-cell derived cancer stemcell, i.e., a stem cell which is capable of giving rise to non-Hodgkin'slymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, mantle celllymphoma or multiple myeloma. Such cells can be readily identified byone of ordinary skill in the art using standard techniques known in theart and those described herein.

The term “immune tolerance,” as used herein, refers to a condition inwhich an animal recognizes a particular cell or antigen(s) as self,which should be recognized as foreign. In other words, the animal'simmune system fails to mount an immune response to a cell or antigen(s)because the antigen is recognized as self instead of foreign. Forexample, the animal fails to mount an immune response against an antigenwhich is specifically expressed on a cancer cell.

The terms “immunoglobulin” and “antibody” (used interchangeably herein)include a protein having a basic four-polypeptide chain structureconsisting of two heavy and two light chains, said chains beingstabilized, for example, by interchain disulfide bonds, which has theability to specifically bind an antigen. The term “single-chainimmunoglobulin” or “single-chain antibody” (used interchangeably herein)refers to a protein having a two-polypeptide chain structure consistingof a heavy and a light chain, said chains being stabilized, for example,by interchain peptide linkers, which has the ability to specificallybind an antigen. The term “domain” refers to a globular region of aheavy or light chain polypeptide comprising peptide loops (e.g.,comprising 3 to 4 peptide loops) stabilized, for example, by β-pleatedsheet and/or intrachain disulfide bond. Domains are further referred toherein as “constant” or “variable,” based on the relative lack ofsequence variation within the domains of various class members in thecase of a “constant” domain, or the significant variation within thedomains of various class members in the case of a “variable” domain.Antibody or polypeptide “domains” are often referred to interchangeablyin the art as antibody or polypeptide “regions.” The “constant” domainsof an antibody light chain are referred to interchangeably as “lightchain constant regions,” “light chain constant domains,” “CL” regions or“CL” domains. The “constant” domains of an antibody heavy chain arereferred to interchangeably as “heavy chain constant regions,” “heavychain constant domains,” “CH” regions or “CH” domains). The “variable”domains of an antibody light chain are referred to interchangeably as“light chain variable regions,” “light chain variable domains,” “VL”regions or “VL” domains). The “variable” domains of an antibody heavychain are referred to interchangeably as “heavy chain constant regions,”“heavy chain constant domains,” “VH” regions or “VH” domains).

Immunoglobulins or antibodies can exist in monomeric or polymeric form,for example, IgM antibodies which exist in pentameric form and/or IgAantibodies which exist in monomeric, dimeric or multimeric form. Otherthan “bispecific” or “bifunctional” immunoglobulins or antibodies, animmunoglobulin or antibody is understood to have each of its bindingsites identical. A “bispecific” or “bifunctional antibody” is anartificial hybrid antibody having two different heavy/light chain pairsand two different binding sites. Bispecific antibodies can be producedby a variety of methods including fusion of hybridomas or linking ofFab′ fragments. See, e.g., Songsivilai & Lachmann, (1990) Clin. Exp.Immunol. 79:315-321; Kostelny et al., (1992) J. Immunol. 148:1547-1553.

The term “antigen-binding portion” of an antibody (or “antibodyportion”) includes fragments of an antibody that retain the ability tospecifically bind to an antigen (e.g., a B-cell specific antigen). Ithas been shown that the antigen-binding function of an antibody can beperformed by fragments of a full-length antibody. Examples of bindingfragments encompassed within the term “antigen-binding portion” of anantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CH1 domains; (ii) a F(ab′)₂ fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the VH and CH1domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Furthermore, although the twodomains of the Fv fragment, VL and VH, are coded for by separate genes,they can be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH regions pair to form monovalent molecules (known as single chainFv (scFv); see e.g., Bird et al., (1988) Science 242:423-426; and Hustonet al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such singlechain antibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. Other forms of single chainantibodies, such as diabodies are also encompassed. Diabodies arebivalent, bispecific antibodies in which VH and VL domains are expressedon a single polypeptide chain, but using a linker that is too short toallow for pairing between the two domains on the same chain, therebyforcing the domains to pair with complementary domains of another chainand creating two antigen binding sites (see e.g., Holliger, P. et al.,(1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J. et al.,(1994) Structure 2:1121-1123). Still further, an antibody orantigen-binding portion thereof may be part of a larger immunoadhesionmolecule, formed by covalent or non-covalent association of the antibodyor antibody portion with one or more other proteins or peptides.Examples of such immunoadhesion molecules include use of thestreptavidin core region to make a tetrameric scFv molecule (Kipriyanov,S. M. et al., (1995) Human Antibodies and Hybridomas 6:93-101) and useof a cysteine residue, a marker peptide and a C-terminal polyhistidinetag to make bivalent and biotinylated scFv molecules (Kipriyanov, S. M.et al., (1994) Mol. Immunol., 31:1047-1058). Antibody portions, such asFab and F(ab')₂ fragments, can be prepared from whole antibodies usingconventional techniques, such as papain or pepsin digestion,respectively, of whole antibodies. Moreover, antibodies, antibodyportions and immunoadhesion molecules can be obtained using standardrecombinant DNA techniques, as described herein. Preferred antigenbinding portions are complete domains or pairs of complete domains.

“Specific binding,” “specifically binds,” “selective binding,” and“selectively binds,” as used herein, mean that the compound, e.g.,antibody or antigen-binding portion thereof, exhibits appreciableaffinity for a particular antigen or epitope and, generally, does notexhibit significant cross-reactivity with other antigens and epitopes.“Appreciable” or preferred binding includes binding with an affinity ofat least 10⁶, 10⁷, 10⁸, 10⁹ M⁻¹, or 10¹⁰ M⁻¹. Affinities greater than10⁷ M⁻¹, preferably greater than 10⁸ M⁻¹ are more preferred. Valuesintermediate of those set forth herein are also intended to be withinthe scope of the present invention and a preferred binding affinity canbe indicated as a range of affinities, for example, 10⁶ to 10¹⁰ M⁻¹,preferably 10⁷ to 10¹⁰ M⁻¹, more preferably 10⁸ to 10¹⁰ M⁻¹. An antibodythat “does not exhibit significant cross-reactivity” is one that willnot appreciably bind to an undesirable entity (e.g., an undesirableproteinaceous entity). For example, in one embodiment, an antibody orantigen-binding portion thereof, that specifically binds to a B-cellspecific antigen, such as, for example, CD-20 or CD-22, will appreciablybind CD-20 or CD-22, but will not significantly react with othernon-CD-20 or non-CD-22 proteins or peptides. Specific or selectivebinding can be determined according to any art-recognized means fordetermining such binding, including, for example, according to Scatchardanalysis and/or competitive binding assays.

The term “humanized immunoglobulin” or “humanized antibody” refers to animmunoglobulin or antibody that includes at least one humanizedimmunoglobulin or antibody chain (i.e., at least one humanized light orheavy chain). The term “humanized immunoglobulin chain” or “humanizedantibody chain” (i.e., a “humanized immunoglobulin light chain” or“humanized immunoglobulin heavy chain”) refers to an immunoglobulin orantibody chain (i.e., a light or heavy chain, respectively) having avariable region that includes a variable framework region substantiallyfrom a human immunoglobulin or antibody and complementarity determiningregions (CDRs) (e.g., at least one CDR, preferably two CDRs, morepreferably three CDRs) substantially from a non-human immunoglobulin orantibody, and further includes constant regions (e.g., at least oneconstant region or portion thereof, in the case of a light chain, andpreferably three constant regions in the case of a heavy chain). Theterm “humanized variable region” (e.g., “humanized light chain variableregion” or “humanized heavy chain variable region”) refers to a variableregion that includes a variable framework region substantially from ahuman immunoglobulin or antibody and complementarity determining regions(CDRs) substantially from a non-human immunoglobulin or antibody.

The term “human antibody” includes antibodies having variable andconstant regions corresponding to human germline immunoglobulinsequences as described by Kabat et al. (See Kabat, et al., (1991)Sequences of proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242).The human antibodies of the invention may include amino acid residuesnot encoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), for example in the CDRs and in particular CDR3. Thehuman antibody can have at least one position replaced with an aminoacid residue, e.g., an activity enhancing amino acid residue which isnot encoded by the human germline immunoglobulin sequence. The humanantibody can have up to twenty positions replaced with amino acidresidues which are not part of the human germline immunoglobulinsequence. In other embodiments, up to ten, up to five, up to three or upto two positions are replaced. In a preferred embodiment, thesereplacements are within the CDR regions as described in detail below.

The term “recombinant human antibody” includes human antibodies that areprepared, expressed, created or isolated by recombinant means, such asantibodies expressed using a recombinant expression vector transfectedinto a host cell, antibodies isolated from a recombinant, combinatorialhuman antibody library, antibodies isolated from an animal (e.g., amouse) that is transgenic for human immunoglobulin genes (see e.g.,Taylor, L. D. et al., (1992) Nucl. Acids Res. 20:6287-6295) orantibodies prepared, expressed, created or isolated by any other meansthat involves splicing of human immunoglobulin gene sequences to otherDNA sequences. Such recombinant human antibodies have variable andconstant regions derived from human germline immunoglobulin sequences(See Kabat E. A., et al., (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242). In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the VH and VLregions of the recombinant antibodies are sequences that, while derivedfrom and related to human germline VH and VL sequences, may notnaturally exist within the human antibody germline repertoire in vivo.In certain embodiments, however, such recombinant antibodies are theresult of selective mutagenesis approach or backmutation or both.

An “isolated antibody” includes an antibody that is substantially freeof other antibodies having different antigenic specificities (e.g., anisolated antibody that specifically binds a B-cell specific antigen andis substantially free of antibodies or antigen-binding portions thereofthat specifically bind other antigens, including other B-cell antigens).An isolated antibody that specifically binds a B-cell specific antigenmay bind the same antigen and/or antigen-like molecules from otherspecies. Moreover, an isolated antibody may be substantially free ofother cellular material and/or chemicals.

The term “chimeric immunoglobulin” or antibody refers to animmunoglobulin or antibody whose variable regions derive from a firstspecies and whose constant regions derive from a second species.Chimeric immunoglobulins or antibodies can be constructed, for exampleby genetic engineering, from immunoglobulin gene segments belonging todifferent species.

II. Exemplary Disorders

Exemplary disorders which may be treated using the methods of theinvention include cancer and in particular, B-cell derived cancers suchas, for example, non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia, mantle cell lymphoma and multiple myeloma.Additional B-cell derived cancers include, for example, B-cellprolymphocytic leukemia, lymphoplasmocytic leukemia, splenic marginalzone lymphoma, marginal zone lymphoma (extra-nodal and nodal), andfollicular lymphoma (e.g., Grade I and II). In general, withoutintending to be bound by theory, it is contemplated that any cancerassociated with the expression of a cancer-specific antigen may betreated using the methods of the invention. In some embodiments, acancer treated using the methods of the present invention is a B-cellderived cancer associated with the expression of one or more B-cellspecific antigens such as, for example, CD3d, CD5, CD6, CD9, CD19, CD20,CD21, CD22, CD23, CD24, CD27, CD28, CD37, CD38, CD40, CD45, CD46, CD48,CD53, CD69, CD70, CD72, CD73, CD79a, CD79b, CD80, CD81, CD83, CD85a,CD85d, CD85e, CD85h, CD85i, CD85j, CD85k, CD86, CD96, CD98, CD100,CD121b, CD124, CD127, CD132, CD150, CD152, CD154, CD157, CD166, CD169,CD179a, CD179b, CD180, CD185, CD196, CD197, CD205, CDw210a, CD213a1,CD257, CD267, CD268, CD269, CD274, CD275, CD276, CD278, CD279, CD300a,CD300c, CD307, CD314, CD316, CD317, CD319, CD320, CDw327, and CD331. Ina particular embodiment, a cancer treated using the methods of theinvention is associated with the expression of CD-20. In anotherembodiment, a cancer treated using the methods of the invention isassociated with the expression of CD-22. In yet another embodiment, acancer treated using the methods of the invention is associated with theexpression of both CD-20 and CD-22.

In some embodiments, a cancer treated using the methods of the inventionis non-Hodgkin's lymphoma or NHL. Non-Hodgkin's lymphoma or NHL, is acancer of the lymphoid tissue which is formed by several types of immunecells including B-cells and T-cells. About 85% of the non-Hodgkin'slymphomas are derived from B-cells. NHL is thought to occur whenB-cells, which produce antibodies, begin to grow abnormally. In someembodiments, non-Hodgkin's lymphoma treated using the methods of theinvention is associated with the expression of CD-20 on B-cells. Inother embodiments, non-Hodgkin's lymphoma is associated with theexpression of CD-22. In yet other embodiments, non-Hodgkin's lymphoma isassociated with the expression of both CD-20 and CD-22.

In some embodiments, a cancer treated using the methods of the inventionis Hodgkin's lymphoma, also referred to as Hodgkin's disease. The cancercells in Hodgkin's disease are called Reed-Sternberg cells, after thetwo doctors who first described them in detail. Under a microscope theylook different from cells of non-Hodgkin's lymphomas and other cancers,and are believed to be a type of malignant B lymphocyte.

In some embodiments, a cancer treated using the methods of the inventionis chronic lymphocytic leukemia (CLL) which is derived from a small Blymphocyte. CLL is mostly found in the blood and in the bone marrow.

In further embodiments, a cancer treated using the methods of theinvention is mantle cell lymphoma.

In yet other embodiments, a cancer treated using the methods of theinvention is multiple myeloma, associated with uncontrolledproliferation of antibody producing cells in the plasma, which developfrom B-cells. Multiple myeloma is the second most common hematologicmalignancy in the United States.

III. Exemplary Oxazaphosphorine Drugs

The methods of the present invention, are based, at least in part, onthe discovery that high-dose pulsed amount of an oxazaphosphorine drugmay be used for breaking immune tolerance, which presents a majorobstacle in the use of immune therapeutics for the treatment of cancersuch as, for example, monoclonal antibodies that bind cancer specificantigens.

Exemplary oxazaphosphorine drugs that may be used in the methods of theinvention include, but are not limited to, for example, cyclophosphamide(CPA), ifosfamide (IFO), and trofosfamide, perfosfamide, or apharmaceutically acceptable salt, solvate, prodrug and metabolitethereof. CPA is widely used in low to intermediate amounts as ananticancer drug, an immunosuppressant, and for the mobilization ofhematopoetic progenitor cells from the bone marrow into peripheral bloodprior to bone marrow transplantation for aplastic anemia, leukemia, andother malignancies. Additional oxazaphosphorine drugs that may be usedin the methods of the invention include, for example, mafosfamide (NSC345842), glufosfamide (D19575, beta-D-glucosylisophosphoramide mustard),NSC 612567 (aldophosphamide perhydrothiazine), and NSC 613060(aldophosphamide thiazolidine).

Both CPA and IFO are prodrugs that require activation by hepaticcytochrome P450 (CYP)-catalyzed 4-hydroxylation, yielding cytotoxicnitrogen mustards capable of reacting with DNA molecules to formcrosslinks and lead to cell apoptosis and/or necrosis. However, morenewly synthesized oxazaphosphorine derivatives such as glufosfamide, NSC612567 and NSC 613060, do not need hepatic activation. They areactivated through other enzymatic and/or non-enzymatic pathways.

In some embodiments according to the present invention, anoxazaphosphorine drug is a lymphocytotoxic but hematopoeitic stem cellsparing high-dose pulsed amount of cyclophosphamide.

IV. Exemplary Antibodies

In various methods of the present invention, cancers derived fromB-cells can be treated using a combination of a high-dose pulsed amountof an oxazaphosphorine drug and a monoclonal antibody which selectivelybinds a B-cell specific antigen.

In some embodiments of the present invention, an antibody is amonoclonal antibody that specifically binds CD-20. In other embodiments,an antibody is a monoclonal antibody that specifically binds CD-22 on aB-cell. However, without wishing to be bound by theory, it iscontemplated that a monoclonal antibody that selectively binds any oneof B-cell specific antigens CD3d, CD5, CD6, CD9, CD19, CD20, CD21, CD22,CD23, CD24, CD27, CD28, CD37, CD38, CD40, CD45, CD46, CD48, CD53, CD69,CD70, CD72, CD73, CD79a, CD79b, CD80, CD81, CD83, CD85a, CD85d, CD85e,CD85h, CD85i, CD85j, CD85k, CD86, CD96, CD98, CD100, CD121b, CD124,CD127, CD132, CD150, CD152, CD154, CD157, CD166, CD169, CD179a, CD179b,CD180, CD185, CD196, CD197, CD205, CDw210a, CD213a1, CD257, CD267,CD268, CD269, CD274, CD275, CD276, CD278, CD279, CD300a, CD300c, CD307,CD314, CD316, CD317, CD319, CD320, CDw327, or CD331, may be used in themethods of the invention. It is also contemplated that any antibody thatresults in depletion or substantial reduction in the number of B-cellsin a sample, or has significant activity in assays for antibodydependent cellular cytotoxicity (ADCC), such as, for example, rituximab,may be used in the methods of the invention.

Commercially available monoclonal antibodies that specifically bindB-cell specific antigens include rituximab, which binds CD-20, andepratuzumab, which binds CD-22.

Antibodies or antigen-binding portions thereof can be tested for bindingto a B-cell or a B-cell specific antigen by, for example, standardassays known in the art, such as ELISA, FACS analysis and/or Biacoreanalysis.

Antibodies or antigen-binding portions useful in the methods of theinvention may be labeled with a detectable substance using well knowntechniques. Suitable detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; and examples of suitable radioactive material include ¹⁴C,¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ^(99m)Tc, ³⁵S or ³H.

IV. Modes Of Administration

The various compounds used in the methods described herein may beadministered orally, parenterally (e.g., intravenously),intramuscularly, sublingually, buccally, rectally, intranasally,intrabronchially, intrapulmonarily, intraperitonealy, topically,transdermally and subcutaneously, for example. The amount of compoundadministered in a single dose may dependent on the subject beingtreated, the subject's weight, the manner of administration and thejudgment of the prescribing physician. Generally, however,administration and dosage and the duration of time for which acomposition is administered will approximate that which are necessary toachieve a desired result.

For example, in some embodiments, a lymphocytotoxic non-myeloablativeamount of a oxazaphosphorine drug used in the methods described hereinis between 100 mg/kg and 200 mg/kg, administered daily from 1 to 7 days.In other embodiments, an effective amount of a lymphocytotoxicnon-myeloablative amount of a oxazaphosphorine drug is between 25 mg/kgand 100 mg/kg, administered daily for 4 consecutive days. In yet otherembodiments, a lymphocytotoxic non-myeloablative amount of aoxazaphosphorine drug is 50 mg/kg administered daily for 4 consecutivedays.

In general, a therapeutically effective amount of a monoclonal antibodysuch as, for example, an antibody that specifically binds CD-20 orCD-22, from about 0.0001 mg/Kg to 0.001 mg/Kg; 0.001 mg/kg to about 10mg/kg body weight or from about 0.02 mg/kg to about 5 mg/kg body weight.In some embodiments, a therapeutically effective amount of a monoclonalantibody is from about 0.001 mg to about 0.01 mg, about 0.01 mg to about100 mg, or from about 100 mg to about 1000 mg, for example.

In some embodiments, an effective amount of an antibody administered toa subject having -Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia or multiple myeloma between about 100 mg/m² and 200mg/m², or between about 200 mg/m² and 300 mg/m² or between about 300 mg/m² and 400 mg/m². In a particular embodiment, an effective amount of amonoclonal antibody that selectively binds a B-cell specific antigen isabout 375 mg/m².

The dose for the oxazaphosphorine drug, e.g., cyclophosphamide, for usein the methods of the present invention can be calculated according tothe ideal body weight of the subject. Ideal body weight can bedetermined, for example, according to Metropolitan Life tables, or anyother standard known in the art. If the patient's actual body weight isless than ideal, the actual weight may be used for the calculation ofthe oxazaphosphorine drug dose.

The optimal pharmaceutical formulations for a desired monoclonalantibody can be readily determined by one or ordinary skilled in the artdepending upon the route of administration and desired dosage. (See, forexample, Remington's Pharmaceutical Sciences, 18th Ed. (1990), MackPublishing Co., Easton, Pa., the entire disclosure of which is herebyincorporated by reference).

Antibodies for use in the methods or compositions described herein canbe formulated for the most effective route of administration, includingfor example, oral, transdermal, sublingual, buccal, parenteral, rectal,intranasal, intrabronchial or intrapulmonary administration.

In some embodiments, the present invention provides kits including oneor more doses of high-dose pulsed amount of an oxazaphosphorine drugand/or one or more doses of an immune therapeutic such as, for example,a B-cell specific monoclonal antibody, packaged with instructions ofuse. Such instructions may pertain to use of the packaged components(i.e., one or more doses of a high-dose pulsed amount of anoxazaphosphorine drug and one or more doses of a B-cell specificmonoclonal antibody) in methods of treating, preventing, ameliorating,eliminating or substantially reducing Hodgkin's lymphoma, non-Hodgkin'slymphoma, chronic lymphocytic leukemia, mantle cell lymphoma or multiplemyeloma, in a patient, or a symptom associated with Hodgkin's lymphoma,non-Hodgkin's lymphoma, chronic lymphocytic leukemia, mantle celllymphoma or multiple myeloma in a patient, by administering the one ormore doses of high-dose pulsed amount of an oxazaphosphorine drug and/orone or more doses of a B-cell specific monoclonal antibody.

Depending on the intended mode of administration, the compounds used inthe methods described herein may be in the form of solid, semi-solid orliquid dosage forms, such as, for example, tablets, suppositories,pills, capsules, powders, liquids, suspensions, lotions, creams, gels,or the like, preferably in unit dosage form suitable for singleadministration of a precise dosage. Each dose may include an effectiveamount of a compound used in the methods described herein in combinationwith a pharmaceutically acceptable carrier and, in addition, may includeother medicinal agents, pharmaceutical agents, carriers, adjuvants,diluents, etc.

Liquid pharmaceutically administrable compositions can prepared, forexample, by dissolving, dispersing, etc., a compound for use in themethods described herein and optional pharmaceutical adjuvants in anexcipient, such as, for example, water, saline aqueous dextrose,glycerol, ethanol, and the like, to thereby form a solution orsuspension. For solid compositions, conventional nontoxic solid carriersinclude, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose,sucrose, magnesium carbonate, and the like. If desired, thepharmaceutical composition to be administered may also contain minoramounts of nontoxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents and the like, for example, sodium acetate,sorbitan monolaurate, triethanolamine sodium acetate, triethanolamineoleate, etc. Actual methods of preparing such dosage forms are known, orwill be apparent, to those skilled in this art; see, for example,Remington's Pharmaceutical Sciences, 18th Ed. (1990), Mack PublishingCo., Easton, Pa., the entire disclosure of which is hereby incorporatedby reference).

V. Methods Of Treatment

Methods of treatment described herein encompass methods of eliminatingor substantially reducing a B-cell derived cancer such as, for example,non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocyticleukemia, mantle cell lymphoma and multiple myeloma. Also encompassedare methods of eliminating immune cells which are capable of elicitingimmune tolerance and methods of depleting cancer stem cells. All methodsdescribed herein exclude the use of stem cell transplantation.

A subject having non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia, mantle cell lymphoma or multiple myeloma can bediagnosed using standard techniques known in the art. For example, adiagnosis may be made by removing a part of a lymph node and examiningthe cells under a microscope. Biopsies may also be taken from other bodytissues.

Subsequent to the diagnosis of a subject as having non-Hodgkin'slymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, mantle celllymphoma or multiple myeloma, the subject can be treated using methodsof the invention, thereby to eliminate or substantially reducenon-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocyticleukemia, mantle cell lymphoma or multiple myeloma in the subject.

In some embodiments, a subject having non-Hodgkin's lymphoma orHodgkin's lymphoma or chronic lymphocytic leukemia or mantle celllymphoma or multiple myeloma is administered a lymphocytotoxic buthematopoeitic stem cell sparing high-dose pulsed amount of anoxazaphosphorine drug, e.g., 50 mg/kg of cyclophosphamide administeredeach day for 4 days. In other embodiments, a subject havingnon-Hodgkin's lymphoma or Hodgkin's lymphoma or chronic lymphocyticleukemia or mantle cell lymphoma or multiple myeloma is administered alymphocytotoxic but hematopoeitic stem cell sparing high-dose pulsedamount of an oxazaphosphorine drug, e.g., 50 mg/kg of cyclophosphamidefor 4 days, followed by a therapeutically effective amount of amonoclonal antibody which specifically binds a B-cell specific antigen,e.g., CD-20 or CD-22.

In some embodiments, a subject having non-Hodgkin's lymphoma, Hodgkin'slymphoma, chronic lymphocytic leukemia, mantle cell lymphoma or multiplemyeloma is administered a monoclonal antibody that selectively binds aB-cell specific antigen prior to the administration of a lymphocytotoxicbut hematopoeitic stem cell sparing high-dose pulsed amount of anoxazaphosphorine drug, e.g., cyclophosphamide. In other embodiments, asubject having non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia, mantle cell lymphoma or multiple myeloma isadministered a monoclonal antibody that selectively binds a B-cellspecific antigen subsequent to the administration of a lymphocytotoxicbut hematopoeitic stem cell sparing high-dose pulsed amount of anoxazaphosphorine drug, e.g., cyclophosphamide. In yet other embodiments,a subject having non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia, mantle cell lymphoma or multiple myeloma isadministered a monoclonal antibody that selectively binds a B-cellspecific antigen both prior and subsequent to the administration of alymphocytotoxic but hematopoeitic stem cell sparing high-dose pulsedamount of an oxazaphosphorine drug, e.g., cyclophosphamide.

In some methods of treatments, according to the invention, a method ofeliminating or substantially reducing non-Hodgkin's lymphoma, Hodgkin'slymphoma, chronic lymphocytic leukemia, mantle cell lymphoma or multiplemyeloma in a subject includes (a) administering a lympocytoxic buthematopoeitic cell sparing high dose pulsed amount of anoxazaphosphorine drug, such that the subject's immune systemreconstitutes without stem cell transplantation; (b) administering atherapeutic amount of an antibody that specifically binds a B-cellspecific antigen; and (c) administering a therapeutic amount of anidiotypic vaccine, thereby to eliminate or substantially reducingnon-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocyticleukemia, mantle cell lymphoma or multiple myeloma in the subject.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification which arehereby incorporated by reference. The embodiments within thespecification provide an illustration of embodiments in this disclosureand should not be construed to limit its scope. The skilled artisanreadily recognizes that many other embodiments are encompassed by thisinvention. All publications and patents cited and sequences identifiedby accession or database reference numbers in this disclosure areincorporated by reference in their entirety. To the extent that thematerial incorporated by reference contradicts or is inconsistent withthe present specification, the present specification will supercede anysuch material. The citation of any references herein is not an admissionthat such references are prior art to the present disclosure.

Unless otherwise indicated, all numbers expressing quantities ofingredients, cell culture, treatment conditions, and so forth used inthe specification, including claims, are to be understood as beingmodified in all instances by the term “about.” Accordingly, unlessotherwise indicated to the contrary, the numerical parameters areapproximations and may vary depending upon the desired properties soughtto be obtained by the present invention. Unless otherwise indicated, theterm “at least” preceding a series of elements is to be understood torefer to every element in the series. Those skilled in the art willrecognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described herein. Such equivalents are intended to beencompassed by the following claims.

1. A method for eliminating or substantially reducing non-Hodgkin'slymphoma in a subject comprising administering a lymphocytotoxic buthematopoeitic stem cell sparing high-dose pulsed amount of anoxazaphosphorine drug to the subject, such that the subject's immunesystem reconstitutes without stem cell transplantation, thereby toeliminate or substantially reduce non-Hodgkin's lymphoma in the subject.2-36. (canceled)